Acetyllysine-recognizing monoclonal antibody and process for producing the same

ABSTRACT

Anti-acetyllysine monoclonal antibody capable of recognizing N ε -acetyllysine regardless of the types of the adjacent amino acids. Namely, a monoclonal antibody having a light chain comprising a constant region having the amino acid sequence represented by SEQ ID NO:1 and a variable region having the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one to several amino acids, and a heavy chain comprising a constant region having the amino acid sequence represented by SEQ ID NO:3 and a variable region having the amino acid sequence represented by SEQ ID NO:4 or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one to several amino acids, and being capable of recognizing N ε -acetyllysine in a protein regardless of the types of the adjacent amino acids, i.e., being capable of accepting adjacent amino acids over a broad range; and a process for producing this monoclonal antibody characterized by using a chemically acetylated protein as an antigen.

TECHNICAL FIELD

[0001] The present invention relates to a novel monoclonal antibody anda method for producing the same. Specifically, the present inventionrelates to a monoclonal antibody capable of recognizing anN^(ε)-acetyllysine residue in a protein without depending on the type ofan adjacent amino acid, and a method for producing the same.

BACKGROUND ART

[0002] In recent years, it has become apparent that an N^(ε)-acetylationof the lysine residue in N-terminal region of a core histone plays animportant role in the control of gene expression of eukaryotes. Theenzyme of a histone acetyltransferase responsible for the acetylationand the enzyme of a histone deacetylase responsible for deacetylationwere cloned in 1996 for the first time, and after that, a plurality ofmolecules having similar activities have been found. In recent years,furthermore, beside histone, it has been found that various kinds ofnon-histone proteins such as p53, TCF, and HMG-1 could be acetylated. Ithas been pointed out that the acetylation may be post-translationmodification that plays various roles as equally as phosphorylation.

[0003] To search for an unknown novel acetylation protein as describedabove, there is no need to discuss the usefulness of a probe moleculethat recognizes an N^(ε)-acetyllysine residue specifically andirrespectively of the adjacent amino acid. An antibody has beenconsidered as the most suitable molecule for the object. However, theantibody capable of recognizing acetyllysine under various conditionsirrespective of the types of the adjacent amino acids has been hardlyreported.

DISCLOSURE OF THE INVENTION

[0004] The present invention has been completed in view of the presentcircumstances and aims to provide an N^(ε)-acetyllysine-recognizinganti-acetyllysine monoclonal antibody which does not particularly dependon the types of the adjacent amino acids and is capable of allowing widevarieties of adjacent amino acids.

[0005] The present inventors have been dedicated to the study of solvingthe above problems and finally attained success to produce ananti-acetyllysine monoclonal antibody capable of allowing wide varietiesof adjacent amino acids. Furthermore, the present invention has beencompleted by determining cDNA sequences of variable regions of therespective monoclonal antibodies being produced to make it clear thatthe produced antibodies have their characterized structures which aresimilar to each other.

[0006] In other words, the present invention relates to a monoclonalantibody that recognizes N^(ε)-acetyllysine. In particular, the presentinvention relates to a monoclonal antibody which does not particularlydepend on the types of the adjacent amino acids and is capable ofallowing wide varieties of adjacent amino acids.

[0007] More specifically, the present invention relates to a monoclonalantibody comprising: (1) a light chain comprising a constant regionhaving the amino acid sequence represented by SEQ ID NO.: 1 and avariable region having the amino acid sequence represented by SEQ IDNO.: 2, or an amino acid sequence derived from this amino acid sequenceby deletion, substitution or addition of one or several amino acids; (2)a heavy chain comprising a constant region having the amino acidsequence represented by SEQ ID NO.: 3 and a variable region having theamino acid sequence represented by SEQ ID NO.: 4, or an amino acidsequence derived from this amino acid sequence by deletion, substitutionor addition of one or several amino acids.

[0008] Furthermore, the present invention relates to a method forproducing the monoclonal antibody characterized in that the chemicallyacetylated protein being is used as an antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows the comparison (ELISA method) between thereactivities of four kinds of anti-acetyllysine monoclonal antibodies ofthe present invention with respect to various acetyllysine-containingpeptides.

[0010]FIG. 2 shows the results of detecting acetylated proteins in thetotal cell lysates of various kinds of cells using four kinds ofanti-acetyllysine monoclonal antibodies.

[0011]FIG. 3 shows the results of investigating whether the ELISAreactivities of four kinds of anti-acetyllysine monoclonal antibodies ofthe present invention are competed with N^(ε)-acetyllysine andN^(α)-acetyllysine.

[0012]FIG. 4 shows the results of comparing between amino acid sequencesof the variable regions of heavy chain and light chain of four kinds ofanti-acetyllysine monoclonal antibodies of the present invention.

BRIEF MODE FOR CARRYING OUT THE INVENTION

[0013] In the monoclonal antibody of the present invention, the variableregion of the light chain having the amino acid sequence, represented bySEQ ID NO.: 2, where one or several amino acids are substitutedincludes, for example, an amino acid sequence of SEQ ID NO.: 5, SEQ IDNO.: 6, or SEQ ID NO.: 7.

[0014] Furthermore, in the monoclonal antibody of the present invention,the variable region of a heavy chain, having the amino acid sequencerepresented by SEQ ID NO.: 4, where one or several amino acids aresubstituted, includes, for example, an amino acid sequence representedby SEQ ID NO.: 8; where one or several amino acids are deleted,includes, for example, an amino acids sequence, represented by SEQ IDNO.: 9; where one or several amino acids are added, includes, forexample, an amino acid sequence represented by SEQ ID NO.: 10,respectively.

[0015] The monoclonal antibodies of the present invention are antibodiesthat recognize N^(ε)-acetyllysine and have the properties of allowingwide varieties of adjacent amino acids without depending on the types oftheir adjacent amino acids when recognizing N^(ε)-acetyllysine residueexisting in protein. These antibodies can be prepared by using variouskinds of acetyllysine-containing molecules as antigens. The antibodyhaving particularly excellent properties is obtained by a method ofpreparation in which a chemically acetylated protein having a pluralityof lysine residues is used as an antigen.

[0016] The antibodies of the present invention include those obtained bybinding synthetic peptides containing acetyllysine with carrier proteinssuch as purple-limpet hemocyanin, or those produced byantibody-producing immortalized cells, obtained by immobilizing theantibody-producing cells such as spleen cells are fused with myelomacells or the like, where the antibody-producing cells are obtained byimmunizing an animal such as a mouse using a protein in which aplurality of lysine residues are chemically acetylated, for examplepurple-limpet hemocyanin acetylated by acetic anhydride, as an antibody.

[0017] Among the above antigen molecules, A method using a proteincontaining a plurality of lysine residues in the molecule thereof ischemically acetylated, is more preferable than a method using a peptidecontaining a single lysine residue as an antigen. Therefore, it iseasily thinkable that using a mixture of various kinds ofacetyllysine-containing peptides is preferable for obtaining excellentantibodies.

[0018] Alternatively, antibody-producing cells can be obtained byvarious methods in which antibody libraries displayed on phages arescreened by means of their affinities with antigens.

[0019] For obtaining such antibody-producing immortalized cells, all themonoclonal antibody producing technologies, which have beenconventionally used, and which will be newly developed in future, can beused.

[0020] The screening of antibody-producing cells can be performed byselecting a clone that produces an antibody allowing adjacent aminoacids as much as possible by using a protein, different from an antigenin which a plurality of lysine residues are acetylated, such asacetylated bovine serum albumin or a acetyllysine-containing peptidehaving various kinds of adjacent amino acids. In addition, the producedantibody molecules can be purified by an affinity column in whichacetyllysine is immobilized or an affinity column using protein A.

[0021] It can be detected whether the produced antibody corresponds tothe antibody of the present invention by analyzing a DNA sequence of thevariable region of the antibody gene in the antibody-producingimmobilized cell, and translating it into a protein, and determined bywhether it has a high similarity of the sequential characteristics ofthe protein with the above sequence or not.

[0022] Furthermore, the present invention provides a gene, preferablyDNA, which encodes a monoclonal antibody that recognizesN^(ε)-acetyllysine of the present invention, which has been mentionedabove. Examples of the DNA of the present invention are shown in SEQ IDNos. 11 to 18 in the sequence table. SEQ ID Nos. 11 to 14 are related tolight chains, and SEQ ID Nos. 15 to 18 are related to heavy chains,respectively. The DNA of the present invention includes complementchains thereof, or the base sequences capable of hybridizing with thesebase sequences under stringent conditions.

[0023] In the monoclonal antibodies of the present invention, assensitized animals, various species of mammals such as mice, rats,rabbits, and dogs, and birds such as chickens can be used. In addition,it is also possible to make chimeric antibodies or human-type antibodiesusing variable regions and/or a hypervariable region of the monoclonalantibody of the present invention.

EXAMPLES

[0024] Hereinafter, the present invention will be explained in detailswith reference to the examples. However, the present invention is notlimited to these examples at all.

Example 1 Preparation of Anti-N^(ε)-Acetyllysine Monoclonal Antibody

[0025] The anti-N^(ε)-acetyllysine monoclonal antibody was prepared witha combination of three kinds of immunological antigens and screeningantigens as described in Table 1. Furthermore, the acetylation of bovineserum albumin and purple-limpet hemocyanin was performed using aceticanhydride by the following method. 10 mg of protein was dissolved in 1ml of a borate buffer (20 mM Na₂B₄O₇, pH 9.3), and then 250 μmol ofacetic anhydride (about 22.6 μl) and 500 μl of 1M NaOH were addedtherein while cooling with ice, followed by incubating for 30 minuteswith stirring occasionally. After the reaction, a solvent was changedusing a G-25 gel filtration (PD-10, Pharmacia Co., Ltd.) and a phosphatebuffer solution (PBS) of acetylated protein was obtained. TABLE 1Immunological antigen Screening antigen Case 1 Conjugate of acetylatedN-terminal Acetylated N-terminal peptide of histone H4 with peptide ofhistone H3 purple-limpet hemocyanin Case 2 Conjugate of acetylatedN-terminal Bovine serum albumin peptide of histone H4 with beingacetylated with purple-limpet hemocyanin acetic anhydride Case 3Purple-limpet hemocyanin being Bovine serum albumin acetylated withacetic anhydride being acetylated with acetic anhydride, and acetylatedpeptide

[0026] The immunization was performed on female Balb/c mice every weekfor three weeks intraperitoneally, using Freund's complete adjuvant atthe first time, and Freund's incomplete adjuvant at the second and thirdtimes. The amount of immunization is 0.1 mg/mouse.

[0027] From the mouse on which the immunization was completed, hybridomathat produces an anti-acetyllysine monoclonal antibody was cloned usingthe conventional method. As a result, Clone-1 from Case 1, Clone-2 fromCase 2, and Clone-3 and Clone-4 from Case 3 were established. Usingvarious kinds of acetyllysine-containing peptides covalently bonded toan ELISA plate (Iwaki Glass Co., Ltd., AquaBind Plate) through theirC-terminal cysteine, the results of comparing reactivities thereon areshown in FIG. 1. In FIG. 1, “A” presents the results of Clone-1, “B”represents those of Clone-2, “C” represents those of Clone-3, and “D”represents those of Clone-4, respectively. The right side of each graphdenotes the concentration of each antibody at the time of providing anabsorbance of 0.5. In addition, a list of peptides used in the figure isas shown in Table 2. As shown in FIG. 1, it was found that each of theantibodies of Clone-1 to Clone-4 showed the binding reactivity toacetyllysine under the conditions in which various kinds of adjacentamino acids were present. From this experiment, three clones, Clone-2 toClone 4, showed their reactivities at almost same level as therespective peptides investigated at this time and it was found that theyaccept adjacent amino acids widely.

[0028] In addition, as a result of determining isotypes of therespective antibodies, it was confirmed that all of them were Ig G1κ.TABLE 2 Name of Peptides Amino Acid Sequence H2A-5-GKQ SGRGK(Ac)QGGKCH2B-5-AKS PEPAK(Ac)SAPAC H2B-12-KKG PAPKK(Ac)GSKKC H2B-15-SKKKKGSK(Ac)KAVTC H3-9-RKS TARK(Ac)STGGKAC H3-14-GKA STGGK(Ac)APRKCH3-18-RKQ KAPRK(Ac)QLATC H3-23-TKA LATK(Ac)AARKSAC H4-5-GKGSGRGK(Ac)GGKGLC H4-16-AKR KGGAK(Ac)RHRKVC H4RC SGRGKGGKGLGKGGAKRHRKVCp53-320 SPQPKK(Ac)KPLC p53-373 HLKSKK(Ac)GQSC p53-382 TSRHKK(A)LMFC

Example 2 Detection of Acetylated Protein By Western Blotting Method

[0029] Five types of cells, B16/BL6, MOLT-4F, HeLa-S3, COS-1, and COS-7were treated with, 1 μM of histone-deacetylase inhibitor, CHAP31, for 24hours, and then cell lysates were prepared and developed on anelectrophoresis, followed by detecting acetylated proteins using theabove four antibodies as primary antibodies. The results were shown inFIG. 2. In FIGS. 2, A, B, C, and D indicate the results of thedetections using Clone-1, Clone-2, Clone-3, and Clone-4, respectively.The concentration of the primary antibodies used for the Westernblotting were 107 ng/ml, 65.7 ng/ml, 258 ng/ml, and 158 ng/ml,respectively, and each of these concentration provide the samereactivity (A492=1) in ELISA where acetylated bovine serum albumin wasimmobilized. The total cell lysates were prepared from the cells (+)after subjecting each cell in the 1 μM CHAP31 treatment for 24 hours orthe cells (−) without the treatment. In each lane, 20 μg of protein wasloaded. Five types of cells being used were 1: B16/BL6, 2: MOLT-4F, 3:HeLa-S3, 4: COS-1, and 5: COS-7, respectively.

[0030] As shown in FIG. 2, the Clone-1 antibody prepared in Case 1 ofExample 1 detected only the acetylation of histone increased by CHAP31.In addition, the Clone-2 antibody prepared in Case 2 detected severalproteins other than histone, but had an insufficient reactivity to theothers. On the other hand, the Clone-3 and Clone-4 antibodies preparedin Case 3 strongly detected acetylated proteins on the position around50 kDa other than histone in MOT-4F, COS-1, and COS-7 cells.Furthermore, it was found that they can detect a plurality of proteinsaround 20 kDa and high-molecular portions in the MOLT-4F cell, and muchmore acetylated proteins in the COS-7 cells. From these results, it wasconfirmed that in the detection of the acetylated non-histone protein bythe Western blotting method, Clone-3 and Clone-4 prepared by usingacetylated purple-limpet hemocyanin as an antigen are particularlyexcellent. In other words, it was found that for preparing the antibodycapable of accepting wide varieties of adjacent amino acids ofacetyllysine, the use of a molecule, of which a plurality of lysineresidues in the protein was acetylated, such as the acetylatedpurple-limpet hemocyanin, as an antibody is preferable.

Example 3 Confirmation of Specificity To N^(ε)-Acetyllysine

[0031] To confirm that the prepared antibody was specifically reactingwith N^(ε)-acetylated lysine, acetylated bovine serum albumin wasimmobilized on an ELISA plate and investigated whether the reactivitiesof the respective antibodies to the ELISA were competed withN^(ε)-acetyllysine and N^(α)-acetyllysine. That is, the ELISA plateimmobilized with a phosphate buffer solution (50 μl) of 1 μg/mlacetylated bovine serum albumin at 4° C. overnight was used, and thenthe inhibitions by N^(ε)-acetyllysine and N^(α)-acetyllysine wereinvestigated under the conditions on which 1 μg/ml of each antibody wasreacted. The results are shown in FIG. 3. In the figure, , ▴, ▪, and ♦show the results of the inhibitions by N^(ε)-acetyllysine and ◯, Δ, □,and ⋄ show the results of the inhibitions by N^(α)-acetyllysine,respectively. Furthermore,  and ◯ are results of the case using Clone-1(AL3D5); ▴ and Δ are results of the case using Clone-2 (AL11); ▪ and □are results of the case using Clone-3 (AKL3H6); and ♦ and ⋄ are resultsof the case using Clone-4 (AKL5C1), respectively.

[0032] As is obvious from FIG. 3, the reactivity of each antibody ofClone-1 to Clone-4 decreased as being competed with N^(ε)-acetyllysine,but not competed with N^(α)-acetyllysine. Accordingly, it has becomeobvious that these antibodies specifically react withN^(ε)-acetyllysine.

Example 4 Determination of cDNA and Amino Acid Sequence in VariableRegion of Each Antibody

[0033] To make clear what kind of amino acid sequence in the variableregion has excellent properties as described in the above examples ofAKL3H6 and AKL5C1 antibodies, the DNA in variable region of the L and Hchains of each antibody using hybridoma was cloned and then the sequencethereof was determined. The cloning was performed by isolating RNA fromthe hybridoma using RNeasy Mini Kit available from QIAGEN Co., Ltd.,performing a reverse transcription reaction using the TrueScript II RTavailable from Sawady Technology using random-9mer as a template, andamplifying the variable-region cDNA by the PCR method, using SuperTaq 2×kit available from Sawady Technology Co., Ltd., using the mix primeravailable from Novagen Co., Ltd. as 5′-primer and5′-ACTGTTCAGGACGCCATTTTGTCGTTCACT-30′ for the light chain and5′-GGATCCAGAGTTCCAGGTCACTGT-3′ for the heavy chain, as 3′-primers. Theresulting DNA fragment was ligated using the DNA Ligation kit ver. 2available from TaKaRa with pT7 Blue T-Vector available from Novagen Co.,Ltd, and then it was used for transforming JM 109 competent cellsavailable from TaKaRa Co., Ltd and seeded in X-gal-, ampicillin-, andIPTG-containing plates, followed by picking up white colonies. Plasmidswere prepared from each of five species of clones that containsnormal-sized inserts, respectively. After that, the DNA sequences weredetermined using an ABI PRISM 310-type automatic sequencer. Thedetermined sequences showed the same sequences as those of five clones,except that a variation which may be caused by a PCR error was found ina part of them, so that these sequences were regarded as the objectiveDNA sequences. The results are shown in SEQ ID NO.: 11 to SEQ ID NO.:18.

[0034] Furthermore, on the basis of the DNA sequences determined asdescribed above, the results of the estimations of the amino acidsequences of the L and H chains are shown in SEQ ID NO.: 19 to SEQ IDNO.: 26, respectively.

[0035] Furthermore, FIG. 4 shows the results of comparing amino acidsequences in the variable regions of the light and heavy chains in therespective antibodies by aligning the amino acid sequences. The aminoacids were represented by means of a one-character notation. Inaddition, the complementarity determining regions in the variable regionwere surrounded by squares and represented by CDR 1 to 3, respectively.

[0036] As is obvious from this figure, each of four kinds of antibodiesobtained by three independent immunologic operations has the commonframework structure. Such a commonality seems to have somethingimportant with the acetyllysine recognition itself. On the other hand,the difference in the properties among the antibodies shown in Examples1 and 2 seems to be attributed to a slight difference in their sequencesdescribed herein; however, since there is no three-dimensionalinformation at present, it is not obvious which portion is responsiblefor the difference in the properties of the antibodies.

[0037] Industrial Applicability

[0038] As the antibody of the present invention can detect acetyllysinewithout depending on the types of adjacent amino acids of acetyllysineso much, it is useful for detecting the state of acetylation ofwell-known various acetylated protein. For instance, it can be easilydetected the change of acetylating level of histone under the influencesof various stimulants by the method such as Western blotting.Furthermore, from the same reason, the antibody of the invention is veryuseful for detecting an unknown novel acetyllysine-containing protein.Concretely, by using an immunoprecipitation method using the antibody ofthe invention; or an affinity column, an antibody chip, or the like, onwhich the antibody of the invention is immobilized, it is expected thatan unknown novel acetyllysine-containing protein would be found.Furthermore, as the antibody of the present invention is a monoclonalantibody, it can be altered to a single-chain antibody by analready-established method. Alternatively, as its epitope isacetyllysine and is small, there is a possibility that either a lightchain or a heavy chain may have its activity, in this case, by means ofa two-hybrid method using DNA encoding this, it can be used fordetecting an acetyllysine-containing protein having a weak-affinity tothe antibody. Furthermore, it may be used in a functional analysis on anacetyllysine-containing protein by expressing in various kinds of cells.Furthermore, if the presence of an acetylated protein having anyconnection with a pathologic condition is revealed in future, it willplay an important role in establishment of a diagnosis method.

1 42 1 57 PRT Mus musculus 1 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val SerIle Phe Pro Pro Ser 1 5 10 15 Ser Glu Gln Leu Thr Ser Gly Gly Ala SerVal Val Cys Phe Leu Asn 20 25 30 Asn Phe Tyr Pro Lys Asp Ile Asn Val LysTrp Lys Ile Asp Gly Ser 35 40 45 Glu Arg Gln Asn Gly Val Leu Asn Ser 5055 2 110 PRT Mus musculus 2 Asp Ala Val Met Thr Gln Thr Pro Leu Ser LeuPro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser SerGln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn Thr Asp Leu Asn Trp Tyr LeuGln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser AsnArg Phe Ser Gly Val Leu 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu GlyVal Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Trp Thr Phe Gly GlyGly Thr Lys Leu Asp 100 105 110 3 54 PRT Mus musculus 3 Gln Gly Thr LeuVal Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser 1 5 10 15 Val Tyr ProLeu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 20 25 30 Thr Leu GlyCys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val 35 40 45 Thr Trp AsnSer Gly Ser 50 4 110 PRT Mus musculus 4 Gln Val Gln Leu Gln Gln Ser GlyAla Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys LysAla Ala Gly Tyr Thr Phe Thr Asn His 20 25 30 Trp Ile Gly Trp Val Lys GlnArg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly SerGly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu ThrAla Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser LeuThr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr TyrGly Ser Trp Phe Ala Tyr Trp Gly 100 105 110 5 110 PRT Mus musculus 5 AspAla Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30Asp Gly Thr Thr Asp Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 7580 Ser Arg Val Glu Ala Glu Glu Leu Gly Val Tyr Phe Cys Leu Gln Val 85 9095 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 110 6110 PRT Mus musculus 6 Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu ProVal Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser GlnSer Leu Glu Lys Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Phe GlnLys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn ArgPhe Ser Gly Val Leu 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly ValTyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly GlyThr Lys Leu Glu 100 105 110 7 110 PRT Mus musculus 7 Asp Ala Val Met ThrGln Thr Pro Leu Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp Gln Ala SerIle Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn ThrTyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu LeuIle Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Leu 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg ValGlu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His ValPro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 110 8 110 PRT Musmusculus 8 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro GlyThr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe ThrLys Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu GluTrp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Thr Asn Tyr Asn GluLys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Ala Asp Pro Ser Ser Thr ThrAla Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala IleTyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asn Tyr Gly Ala Trp Phe Ala Tyr TrpGly 100 105 110 9 109 PRT Mus musculus 9 Gln Val Gln Leu Gln Gln Ser GlyAla Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys LysAla Ala Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Ile Gly Trp Val Lys GlnArg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ala GlyGly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu ThrAla Asp Thr Ser Ser Ser Thr Ile Tyr 65 70 75 80 Met Gln Leu Ser Ser LeuThr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Cys Tyr Gly Tyr Gly GlyAla Trp Phe Ser Tyr Trp Gly 100 105 10 112 PRT Mus musculus 10 Gln ValGln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 SerVal Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 TrpIle Gly Trp Val Lys Gln Arg Pro Gly His Asp Leu Glu Trp Ile 35 40 45 GlyAsp Ile Tyr Pro Gly Ser Gly Tyr Ala Tyr Tyr Asn Glu Lys Phe 50 55 60 LysGly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Phe Tyr Tyr Cys 85 90 95Val Arg Ser Tyr Phe Ala Asp Gly Pro Ala Trp Phe Ala Tyr Trp Gly 100 105110 11 501 DNA Mus musculus 11 gatgctgtga tgacccaaac tccactctccctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca ggtctagtca gagccttgaaaacagtaatg gaaacaccga tttgaactgg 120 tacctccaga aaccaggcca gtctccacagctcctgatct acagggtttc caaccgattt 180 tctggggtcc tagacaggtt cagtggtagtggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtttatttctgcc tccaagttac acatgtcccg 300 tggacgttcg gtggaggcac caagctggacatcaaacggg ctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagttaacatctggag gtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaatgtcaagtgga agattgatgg cagtgaacga 480 caaaatggcg tcctgaacag t 501 12 501DNA Mus musculus 12 gatgctgtga tgacccaaac tccactctcc ctgcctgtcagtcttggaga tcaagcctcc 60 atctcttgca ggtctagtca gagccttgaa aacagtgatggaaccaccga tttgaactgg 120 tacctccaga aaccaggcca gtctccacag ctcctgatctacagggtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggtagt ggatcagggacagatttcac actgaaaatc 240 agcagagtgg aggctgagga attgggagtt tatttctgccttcaagttac acatgtcccg 300 tggacgttcg gtggaggcac caagctggaa atcaaacgggctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggaggtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtggaagattgatgg cagtgaacga 480 caaaatggcg tcctgaacag t 501 13 501 DNA Musmusculus 13 gatgctgtga tgacccaaac tccactctcc ctgcctgtca gtcttggagatcaagcctcc 60 atctcttgca ggtctagtca gagccttgaa aaaagtaatg gaaacacctatttgaactgg 120 tatttccaga aaccaggcca gtctccacag ctcctgatct acagggtttccaaccgattt 180 tctggggtcc tagacaggtt cactggtagt ggatcaggga cagatttcacattgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tatttctgcc tccaagttacacatgtcccg 300 tggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgcaccaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagtcgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatggcagtgaacga 480 caaaatggcg tcctgaacag t 501 14 501 DNA Mus musculus 14gatgctgtga tgacccaaac tccactctcc ctgtctgtca gtcttggaga tcaagcctcc 60atctcttgca ggtctagtca gagccttgaa aacagtaatg gaaacaccta tttgaactgg 120tacctccaga aaccaggcca gtctccacag ctcctgatct acagggtttc caaccgattt 180tctggggtcc tagacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga tttgggagtt tatttctgcc tccaagttac acatgtcccg 300tggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta 360tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480caaaatggcg tcctgaacag t 501 15 492 DNA Mus musculus 15 caggtccagctgcagcagtc tggagctgag ttggtaaggc ctgggacttc agtgaagatg 60 tcctgcaaggctgctggata caccttcact aaccactgga taggttgggt aaagcagagg 120 cctggacatggccttgagtg gattggagat atttaccctg gaagtggtta tactaactac 180 aatgagaagttcaagggcaa ggccacactg actgcagaca catcctccag cacagcctac 240 atgcagctcagcagcctgac atctgaggac tctgccatct attactgtgc aagatccgat 300 tactacggctcctggtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc 360 aaaacgacacccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 420 atggtgaccctgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 480 aactctggat cc492 16 492 DNA Mus musculus 16 caggtccagc tgcagcagtc tggagctgagctggtaaggc ctgggacttc agtgaagatg 60 tcctgcaagg ctgctggata caccttcactaaatactgga taggttgggt aaagcagagg 120 cctggacatg gccttgagtg gattggagatatttaccctg gaagtggtta tactaactac 180 aatgagaaat tcaagggcaa ggccaaactgactgcagacc cttcctccac cacagcctac 240 ctgcagctca gcagcctgac atctgaggactctgccatct attactgtgc aagagcggga 300 aattacggcg cctggtttgc ttactggggtcaagggactc tggtcactgt ctctgcagcc 360 aaaacgacac ccccatctgt ctatccactggcccctggat ctgctgccca aactaactcc 420 atggtgaccc tgggatgcct ggtcaagggctatttccctg agccagtgac agtgacctgg 480 aactctggat cc 492 17 489 DNA Musmusculus 17 caggtccagc tgcagcagtc tggagctgag ctggtaaggc ctgggacttcagtgaagatg 60 tcctgcaagg ctgctggata caccttcact aagtattgga taggttgggttaagcagagg 120 cctggacatg gccttgagtg gattggagat atttaccctg caggtggttatactaactac 180 aatgagaagt tcaagggcaa ggccacactg actgcagaca catcctccagcacaatctac 240 atgcagctca gcagcctgac atctgaggac tctgccatct attactgttgctatggttac 300 ggcggggcct ggttttctta ctggggccaa gggactctgg tcactgtctctgcagccaaa 360 acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaactaactccatg 420 gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagtgacctggaac 480 tctggatcc 489 18 498 DNA Mus musculus 18 caggtccaactgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaagatg 60 tcctgcaaggctgctggata caccttcact aactactgga taggttgggt aaagcagagg 120 cctggacatgaccttgagtg gattggagat atttaccctg gaagtggtta tgcttactac 180 aatgagaagttcaagggcaa ggccacactg actgcagaca catcctccag cacagcctac 240 atgcagctcagcagcctgac atctgaggac tctgccttct attactgtgt aagatcctac 300 ttcgctgatggcccggcctg gtttgcttac tggggccaag ggactctggt cactgtctct 360 gcagccaaaacgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 420 aactccatggtgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 480 acctggaactctggatcc 498 19 167 PRT Mus musculus 19 Asp Ala Val Met Thr Gln Thr ProLeu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser CysArg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn Thr Asp Leu AsnTrp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr ArgVal Ser Asn Arg Phe Ser Gly Val Leu 50 55 60 Asp Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala GluAsp Leu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Trp ThrPhe Gly Gly Gly Thr Lys Leu Asp Ile Lys 100 105 110 Arg Ala Asp Ala AlaPro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr SerGly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro LysAsp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 GlnAsn Gly Val Leu Asn Ser 165 20 167 PRT Mus musculus 20 Asp Ala Val MetThr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln AlaSer Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asp Gly ThrThr Asp Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln LeuLeu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser ArgVal Glu Ala Glu Glu Leu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr HisVal Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 ArgAla Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145150 155 160 Gln Asn Gly Val Leu Asn Ser 165 21 167 PRT Mus musculus 21Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 1015 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Lys Ser 20 2530 Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Phe Gln Lys Pro Gly Gln Ser 35 4045 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Leu 50 5560 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 7075 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val 8590 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser SerGlu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu AsnAsn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp GlySer Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser 165 22 167 PRTMus musculus 22 Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val SerLeu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser LeuGlu Asn Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys ProGly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe SerGly Val Leu 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrLeu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr PheCys Leu Gln Val 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr LysLeu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile PhePro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val ValCys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys TrpLys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser165 23 164 PRT Mus musculus 23 Gln Val Gln Leu Gln Gln Ser Gly Ala GluLeu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala AlaGly Tyr Thr Phe Thr Asn His 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg ProGly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Ser Gly TyrThr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala AspThr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Tyr Gly SerTrp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser AlaAla Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly SerAla Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val LysGly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser GlySer 24 164 PRT Mus musculus 24 Gln Val Gln Leu Gln Gln Ser Gly Ala GluLeu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala AlaGly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg ProGly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Ser Gly TyrThr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Ala AspPro Ser Ser Thr Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asn Tyr Gly AlaTrp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser AlaAla Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly SerAla Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val LysGly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser GlySer 25 163 PRT Mus musculus 25 Gln Val Gln Leu Gln Gln Ser Gly Ala GluLeu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala AlaGly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg ProGly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Ala Gly Gly TyrThr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala AspThr Ser Ser Ser Thr Ile Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Cys Tyr Gly Tyr Gly Gly Ala TrpPhe Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala AlaLys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser AlaAla Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys GlyTyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser26 166 PRT Mus musculus 26 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu LeuVal Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala GlyTyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Arg Pro GlyHis Asp Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Ser Gly Tyr AlaTyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp ThrSer Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser GluAsp Ser Ala Phe Tyr Tyr Cys 85 90 95 Val Arg Ser Tyr Phe Ala Asp Gly ProAla Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val SerAla Ala Lys Thr Thr Pro Pro Ser 115 120 125 Val Tyr Pro Leu Ala Pro GlySer Ala Ala Gln Thr Asn Ser Met Val 130 135 140 Thr Leu Gly Cys Leu ValLys Gly Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Thr Trp Asn SerGly Ser 165 27 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 27 Ser Gly Arg Gly Lys Xaa Gln Gly Gly LysCys 1 5 10 28 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 28 Pro Glu Pro Ala Lys Xaa Ser Ala Pro AlaCys 1 5 10 29 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 29 Pro Ala Pro Lys Lys Xaa Gly Ser Lys LysCys 1 5 10 30 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 30 Lys Lys Gly Ser Lys Xaa Lys Ala Val ThrCys 1 5 10 31 12 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 31 Thr Ala Arg Lys Xaa Ser Thr Gly Gly LysAla Cys 1 5 10 32 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 32 Ser Thr Gly Gly Lys Xaa Ala Pro Arg LysCys 1 5 10 33 11 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 33 Lys Ala Pro Arg Lys Xaa Gln Leu Ala ThrCys 1 5 10 34 12 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 34 Leu Ala Thr Lys Xaa Ala Ala Arg Lys SerAla Cys 1 5 10 35 12 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 35 Ser Gly Arg Gly Lys Xaa Gly Gly Lys GlyLeu Cys 1 5 10 36 12 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 36 Lys Gly Gly Ala Lys Xaa Arg His Arg LysVal Cys 1 5 10 37 22 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 37 Ser Gly Arg Gly Lys Gly Gly Lys Gly LeuGly Lys Gly Gly Ala Lys 1 5 10 15 Arg His Arg Lys Val Cys 20 38 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide38 Ser Pro Gln Pro Lys Lys Xaa Lys Pro Leu Cys 1 5 10 39 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide39 His Leu Lys Ser Lys Lys Xaa Gly Gln Ser Cys 1 5 10 40 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide40 Thr Ser Arg His Lys Lys Xaa Leu Met Phe Cys 1 5 10 41 30 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer41 actgttcagg acgccatttt gtcgttcact 30 42 24 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 42 ggatccagagttccaggtca ctgt 24

1-2. (canceled)
 3. A monoclonal antibody recognizing anN^(ε)-acetyllysine, comprising: (1) a light chain comprising a constantregion having an amino acid sequence represented by SEQ ID NO. 1 and avariable region having an amino acid sequence represented by SEQ ID No.2 or an amino acid sequence derived from this amino acid sequence bydeletion, substitution, or addition of one or several amino acids; and(2) a heavy chain comprising a constant region having an amino acidsequence represented by SEQ ID No. 3 and a variable region having anamino acid sequence represented by SEQ ID No. 4 or an amino acidsequence derived from this amino acid sequence by deletion,substitution, or addition of one or several amino acids.
 4. Themonoclonal antibody according to claim 3, wherein the variable region ofthe light chain comprises an amino acid sequence derived from the aminoacid sequence, represented by SEQ ID No. 2, by substitution of one orseveral amino acids.
 5. The monoclonal antibody according to claim 4,wherein the amino acid sequence derived from the amino acid sequence,represented by SEQ ID No. 2, by substitution of one or several aminoacids is an amino acid sequence represented by SEQ ID No.
 5. 6. Themonoclonal antibody according to claim 4, wherein the amino acidsequence derived from the amino acid sequence, represented by SEQ ID No.2, by substitution of one or several amino acids is an amino acidsequence represented by SEQ ID No.
 6. 7. The monoclonal antibodyaccording to claim 4, wherein the amino acid sequence derived from theamino acid sequence, represented by SEQ ID No. 2, by substitution of oneor several amino acids is an amino acid sequence represented by SEQ IDNo.
 7. 8. The monoclonal antibody according to any of claims 3 to 7,wherein the variable region of the heavy chain comprises an amino acidsequence derived from the amino acid sequence, represented by SEQ ID No.4 by deletion, substitution, or addition of one or several amino acids.9. The monoclonal antibody according to claim 8, wherein the amino acidsequence derived from the amino acid sequence, represented by SEQ ID No.4 by substitution of one or several amino acids is an amino acidsequence represented by SEQ ID No.
 8. 10. The monoclonal antibodyaccording to claim 8, wherein the amino acid sequence derived from theamino acid sequence, represented by SEQ ID No. 4, by deletion of one orseveral amino acids is an amino acid sequence represented by SEQ ID No.9.
 11. The monoclonal antibody according to claim 8, wherein the aminoacid sequence derived from the amino acid sequence, represented by SEQID No. 4, by addition of one or several amino acids is an amino acidsequence represented by SEQ ID No.
 10. 12. The monoclonal antibodyaccording to Claim 3, wherein the monoclonal antibody is a mousemonoclonal antibody.
 13. A method for producing a monoclonal antibodyaccording to claim 3, wherein a chemically acetylated protein is used asan antigen.
 14. The production method according to claim 13, wherein theprotein is a protein having a plurality of lysines in the molecule. 15.The production method according to claim 13, wherein the chemicallyacetylated protein is acetylated purple-limpet hemocyanin.
 16. Themonoclonal antibody according to claim 3, capable of allowing widevarieties of adjacent amino acids without specifically depending on thetypes of adjacent amino acids at the time of recognizing theN^(ε)-acetyllysine in a protein.
 17. A gene encoding the monoclonalantibody of claim 3 wherein the monoclonal antibody recognizesN^(ε)-acetyllysine.
 18. The gene according to claim 17 wherein the genecomprises a base sequence represented by any of SEQ ID NO.:11 to
 14. 19.The gene according to claim 17 wherein the gene comprises complementchains of a base sequence represented by any of SEQ ID NO.: 11 to 14 orthe base sequence capable of hybridizing with these base sequences underthe stringent conditions.
 20. The gene according to claim 17 wherein thegene comprises a base sequence represented by any of SEQ ID NO.: 15 to18.
 21. The gene according to claim 17 wherein the gene comprisescomplement chains of a base sequence represented by any of SEQ ID NO.:15 to 18 or the base sequence capable of hybridizing with these basesequences under the stringent conditions.